The delivery of biological material into cells, especially the delivery of DNA, raises many promising opportunities to treat diseases of both genetic and infectious origin. It is based on delivering biologically active substances such as peptides, proteins or nucleic acids % into somatic cells of an organism in order to influence the cell metabolism or switch off defective genes, to replace a defective gene with an intact gene, or to enable these cells to form a protein that possesses a prophylactic or therapeutic effect.
Examples of genetically caused diseases in which gene therapy represents a promising approach are numerous. Other possible applications are in immune regulation, in which immunity is achieved by the administration of functional nucleic acid codes for a secreted protein antigen or for a non-secreted protein antigen, by immunisation. Other examples of genetic defects in which a nucleic acid which codes for the defective gene can be administered, e.g. in a form individually tailored to the particular requirement, include muscular dystrophy (dystrophin gene), cystic fibrosis (CFTR gene), hyper-holesterolemia (LDL receptor gene). Gene therapy methods of treatment are also potentially of use when hormones, growth factors or proteins with a cytotoxic or immune-modulating activity are to be synthesised in the body.
Gene therapy also appears promising for the treatment of cancer by administering so-called “cancer vaccines”. In order to increase the immunogenicity of tumor cells, they are altered to render them either more antigenic or to make them produce certain cytokines in order to trigger an immune response. This is accomplished by transfecting the cells with DNA coding for a cytokine, e.g. IL-2, IL4, IFN gamma, TNF alpha and others. To date, gene transfer into autologous tumor cells has been accomplished via retroviral vectors as therapeutic agents for blocking the expression of certain genes (such as deregulated oncogenes or viral genes) in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells and exert their inhibiting effect therein, even if their intracellular concentration is low, caused by their restricted uptake by the cell membrane as a result of the strong negative charge of the nucleic acids.
Given the substantial benefits that may accrue from the delivery there is a clear need for safe and efficient biological material, mainly nucleic acids, delivery systems.
Various techniques are known for gene transfer into mammalian cells in vitro, e.g. introducing of DNA by means of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran or calcium phosphate precipitation methods. Cationic lipids have been successfully used to transfer DNA. The cationic component of such lipids can compact DNA in solution. This method has been shown to result in heavily aggregated DNA complexes that, when used for transfecting the DNA in vitro, results in increased efficiency of gene transfer and expression (relative to naked DNA). Although the formation of these complexes can promote gene transfer in vitro, the injection of such complexes in vivo does not result in long lasting and efficient gene transfer.
Currently, viruses comprise the most popular vectors for in vitro and in vivo gene delivery. These vectors have been developed to bring about the transfer of genes by using the efficient entry mechanisms of their parent viruses. This strategy was used in the construction of recombinant retroviral and adenoviral vectors in order to achieve a highly efficient gene transfer in vitro and in vivo. For all their efficiency, these vectors are subject to restrictions in terms of the size and construction of the DNA which is transferred and there is a danger that viral coat proteins can trigger immune reactions in the recipient. Furthermore, these agents constitute safety risks in view of the co-transfer of viable viral gene elements of the original virus. Thus, for example, the use of retroviruses is problematic because it involves, at least to a small percentage, the danger of side effects such as infection with the virus (by recombination with endogenous viruses and possible subsequent mutation into pathogenic form) or the formation of cancer. Moreover, the stable transformation of the somatic cells of the patient, as achieved by means of retroviruses, is not desirable in each case because this can only make the treatment more difficult to reverse, e.g. if side effects occur.
Recently, synthetic vectors have been suggested as an alternative to viruses and alternative strategies for gene transfer have been developed.
One example of this is the transfer of genes into the cell via the extremely efficient route of receptor-mediated endocytosis (Ref.1,2). This approach uses bifunctional molecular conjugates which have a DNA binding domain and a domain with specificity for a cell surface receptor. A DNA-binding moiety is usually poly-L-lysine(PLL). Complexes are formed with DNA through electrostatic interactions between the positively charged lysine residues and the negatively charged phosphates in the DNA backbone. The efficiency of gene expression achieved by receptor-mediated endocytosis is affected by a variety of factors, including the diameter of the complexes and the type of targeting ligand used.
There is generally accepted notion that for successful application in vivo the DNA delivery system must be small enough to gain access to target cells. This frequently involves extravasation through endothelia, and the hyperpermeable endothelia associated with tumors have a size restriction of about 70 nm (Ref.3). In addition, most forms of triggered membrane penetration act via the endosomal membrane following endocytosis and pinocytic internalisation is usually limited to materials of less than 100 nm diameter. It has been shown that nucleic acid compaction rather than surface charge was critical for efficient nuclear trafficking(Ref.4).
Given the large size of biomolecules, especially of DNA expression vectors in free solution, it is advantageous for the DNA to be compressed. It is known that DNA can be condensed into polyelectrolyte complexes simply by the addition of polycations such as polylysine (PLL). It is known that conjugates containing higher molecular weight (mw) PLL clearly have on average a larger size and greater polydispersity than those containing lower molecular weight PLL. For example, conjugates based on the largest PLL (224500 Da) show a broad polydispersity of size, ranging up to maximum diameters of 300 nm, while conjugates based on the smallest PLL (3970 DA) show a small size and relatively uniform distribution (diameter ranging from 20–30 nm) (Ref. 5).
Polycations are known to exert a range of non-specific toxicity effects and the concentration of electrostatic charges resulting from polyelectrolyte condensation could yield particles with extremely high charge density and possibly even increased toxicity. The conjugates formed using higher molecular weight PLL show considerably greater cytotoxicity than those formed with the lowest molecular weight polycations (Ref.5).
In DNA-PLL complexes, the function of PLL is to condense DNA into a compact structure. One of the most effective DNA condensing agents is spermine, a tetramine. A peptide analogue of spermine was synthesised to test the premise that short synthetic peptides could in fact function as well or better than PLL. The peptide K8 (Ref.6) is a superior replacement for the high molecular weight PLL. The potential cytotoxicity was compared with that of PLL. K8 is at least 1000-fold less toxic than PLL for HepG2 cells. Similar results were obtained in other cell lines.
Previous studies with DNA-PLL complexes have demonstrated that an endosomal lysis agent is necessary for high efficiency gene transfer. A replication defective adenovirus has been frequently used to achieve levels of expression comparable to recombinant adenovirus containing the same exogenous gene. It is well known that the host immune response to adenovirus limits its use to a single administration. To replace adenovirus as an endosomal lytic agent, fusion peptide of virus protein has been employed. JTS-1, a novel amphipatic peptide has been created (Ref.6). High levels of gene expression were achieved in a variety of cell line's, indicating that DNA-K8/JTS-1 complexes are highly useful for gene delivery in vitro.
The data demonstrate that it is feasible to construct simple DNA-peptide complexes that give high efficiency gene: delivery into cultured cells. These complexes contain only three components: DNA, condensing peptide, and lytic peptide, all of which are molecularly defined and easily undergo self-assembly into an active DNA delivery system. Future development of these complexes holds promise of replacing viral vectors, however, their in vivo application is yet unknown.
Recently (Ref.7) the cationic polymer polyethylenimine (PEI) was shown to mediate efficient gene transfer into a variety of cells without the addition of any cell-binding ligand or endosomolytic moiety. This compound, in contrast to PLL, combines DNA binding and condensing activity with a high pH-buffering capacity. Every third atom of the PEI backbone is protonatable amino nitrogen atom making the polymer an efficient “proton sponge”. Endosomal and lysosomal buffering is considered to protect DNA from degradation and to promote release from the acidic vesicles. These properties make PEI a very attractive DNA-binding core for more sophisticated vectors containing cell-binding domains and other cell entry functions. It has been shown that the ligand-PEI conjugates can mediate efficient transfection of cultured tumor cells in a receptor-ligand-dependent manner. This findings indicate that ligand-conjugated PEI might be promising vector for receptor-specific gene delivery (Ref.8).
PEI-DNA complexes with different ratios of PEI nitrogen to DNA phosphate (N/P ratio) have been prepared and tested in a variety of in vivo models. Earlier experiments carried out with the branched 25 kDa PEI show this polymer to be toxic, causing death within a few minutes, even when used at low N/P ratios. Better results are obtainable with linear polymers with a mean molecular weight of 22 kDA (Exgene 500). When complexing a reporter gene (pCMV-Luc) with Exgene 500 at ratios of 3 to 5 (N/P), transgene expression may be found 24 h later in lungs, heart, spleen, liver, kidney and brain (Ref.9). However, toxic and immunogenic characteristics may probably riot be overcome with the use of PEI.
Electroporation, another non-viral delivery system, is used to deliver biological material into target cells by applying an electrical field as described in U.S. Pat. No. 4,849,355; and U.S. Pat. No. 5,232,856. To deliver biological material into cells electric pulses are applied to target cells f.e. in a cell-suspension. The biological material in the suspension may diffuse into the cell through small pores, which are formed in the cell membrane by the application of the electric pulses.
Liposomal techniques have been combined with electroporation techniques to encapsulate the biological materials in liposomes and fuse the liposomes with targeted cells by electrofusion in order to achieve higher efficiency delivery. However, the liposome are weakly loaded and do not fuse well with the target cell in the electrical field.
In the U.S. Pat. No. 5,789,213, fully incorporated by reference, an electroporation system is described that relates to the use of a two phase polymer system that concentrates biological materials with the target cells, such that the materials can be introduced into target cells during and after administration of an electric pulse by concentrating both the target cells, and biological materials to be loaded, into one of the two phases.
A two-phase polymer method is capable of separating or partitioning cells, proteins and minerals (described in U.S. Pat. No. 4,181,589; and Partitioning in Aqueous Two-Phase Systems, 1985, eds., H. Walter, D. Brooks, and D. Fisher, pubis. Academic Press wherein polymer concentrations are % w/w unless-noted otherwise). The partition of particles into different-polymer phases depends on the interfacial energy of the particles and the polymer solutions. By varying the interfacial energy governed by the polymer and salt concentrations, selected particles (cells, macromolecules) can be driven into a given phase, hence achieving the purpose of separation or partitioning by the use of combinations of polymers.
According, to U.S. Pat. No. 5,789,213 a composition is used which functions to concentrate both target cells and biological materials into a single phase, and function to reduce the volume of this phase by osmotic control so that cells and biological materials are encapsulated in this single phase in a concentrated form during electroporation. Biological materials are then driven into the target cells during electroporation, and subsequent colloidal-osmotic swelling of cells after electroporation is limited, result in a higher loading efficiency. For example a two-phase polymer system using polyethylene glycol (PEG; molecular size (m.w,) 8,000 (in daltons)) and one of three formulations of dextran (dx; m.w. 9,000, and 71,000 and 249,000) is described.
However, electroporation beyond other disadvantages requires special equipment and is limited to use in vitro.
Delivery of bioactive molecules such as nucleic acid can be significantly enhanced by immobilisation of the bioactive molecule in a polymer microparticles which facilitates transfer of the molecule into the targeted areas as described above. However, polymers preferably must be non-toxic, containing no toxic monomers and degrading into non-toxic components, be biocompatible, be chemically compatible with the substances to be delivered. To make microparticles from synthetic and natural polymers a number of different techniques have been developed.
In the U.S. Pat. No. 5,849,884, fully incorporated by reference, macromolecular microparticles and a method of production and use are described. This method is based on the collapse of macromolecules with a tertiary or quaternary structure, forming the basic structure: elements. Microparticles are produced by mixing macromolecules in solution or a liquid phase with a polymer or mixture of polymers in solution or a liquid phase in the presence of an energy source for a sufficient amount of time to form particles. The solution is preferably an aqueous solution. Either the macromolecule solution is added to the polymer or the polymer solution is added to the macromolecule solution to cause removal of water from, or dehydration of the macromolecules. This process is also referred to by those skilled in the art as “volume exclusion”.
The types of macromolecules forming the microparticles include proteins, peptides, carbohydrates, conjugates, nucleic acids, viruses, or mixtures thereof. Since macromolecules are the major structure forming elements within the microparticles suitable macromolecules need to have or to be capable of having a tertiary or quaternary structure. The preferred polymer is polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), dextran (Dx), polyoxyethylene-polyoxypropylene copolymer (PPC), polyvinyl alcohol (PVA), or mixtures thereof.
Each microparticle is composed of at least 40% by weight macromolecules and less or equal than 30% by weight polymer molecules, which are intertwined or interspersed in the microparticle and are generally homogeneously distributed.
In order to induce the microparticles forming collapse of the macromolecules the macromolecule-polymer solution is incubated in the presence of an energy source for a predetermined length of time. The preferred energy source is heat. However, possible energy sources include heat, radiation, and ionisation, alone or in combination with sonification, vortexing, mixing or stirring. Preferably, the macromolecule-polymer solution mixture is incubated in a water bath at a temperature greater than or equal to 37° C. and less than or equal 90° C. for between approximately 5 minutes and 2 hours. Most preferably, the mixture is incubated for 5–30 minutes at a temperature between 50 and 90° C.
This method needs the macromolecule-polymer solution to be adjusted to a certain pH range, either before, after or during the mixing of the polymer with the macromolecule, to a pH near the isoelectric point (pl) of the macromolecule, preferably within 3 to 4 pH units of the pl of the macromolecule; most preferably within 1.5 to 2 pH units of the pl of the macromolecule.
Microparticles composed of nucleic acids have to be prepared by first mixing the nucleic acid either with a protein, such as bovine serum albumin, or, because nucleic acids are anions, the addition of a cation, such as poly-L-lysine (PLL), which aids greatly in the formation of microparticles.
In respect to this composition it is disadvantageous, that the method is limited to a certain pH range of the macromolecule polymer solution. Additionally, it requires a special equipment in order to incubate the solution with energy, for example heating, in combination with stirring, vortexing or mixing.
Further more, it is limited to macromolecules having or capable of having a tertiary or quaternary structure.
Most of all, this method does not solve the difficulties related to the transfer of genes into cells of an organism. In particular, the problems related to the penetration of biological material, especially nucleic acid, into cells and their nuclei are not solved.